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Infection with canine heartworm ( Dirofilaria immitis ), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di -CPI from D. immitis . Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di -CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-r Di -CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di -CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di -CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di -CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di -CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis in dogs, cats and occasionally in humans. Prevention of the disease has been mainly by monthly use of the macrocyclic lactone (ML) endectocides during the mosquito transmission season. Recently, ML resistance has been confirmed in D. immitis and therefore, there is a need to find new classes of anthelmintics. One of the mechanisms associated with ML resistance in nematodes has been the possible role of ATP binding cassette (ABC) transporters in reducing drug concentrations at receptor sites. ABC transporters, mainly from sub-families B, C and G, may contribute to multidrug resistance (MDR) by active efflux of drugs out of the cell. Gene products of ABC transporters may thus serve as the targets for agents that may modulate susceptibility to drugs, by inhibiting drug transport. ABC transporters are believed to be involved in a variety of physiological functions critical to the parasite, such as sterol transport, and therefore may also serve as the target for drugs that can act as anthelmintics on their own. Knowledge of polymorphism in these ABC transporter genes in nematode parasites could provide useful information for the process of drug design. We have identified 15 ABC transporter genes from sub-families A, B, C and G, in D. immitis, by comparative genomic approaches and analyzed them for polymorphism. Whole genome sequencing data from four ML susceptible (SUS) and four loss of efficacy (LOE) pooled populations were used for single nucleotide polymorphism (SNP) genotyping. Out of 231 SNPs identified in those 15 ABC transporter genes, 89 and 75 of them were specific to the SUS or LOE populations, respectively. A few of the SNPs identified may affect gene expression, protein function, substrate specificity or resistance development and may be useful for transporter inhibitor/anthelmintic drug design, or in order to anticipate resistance development., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dirofilaria immitis, a filarial parasite, causes cardiopulmonary dirofilariasis in dogs, cats and wild canids. The macrocyclic lactone (ML) class of drugs has been used to prevent heartworm infection. There is confirmed ML resistance in D. immitis and thus there is an urgent need to find new anthelmintics that could prevent and/or control the disease. Targeting ion channels of D. immitis for drug design has obvious advantages. These channels, present in the nematode nervous system, control movement, feeding, mating and respond to environmental cues which are necessary for survival of the parasite. Any new drug that targets these ion channels is likely to have a motility phenotype and should act to clear the worms from the host. Many of the successful anthelmintics in the past have targeted these ion channels and receptors. Knowledge about genetic variability of the ion channel and receptor genes should be useful information for drug design as receptor polymorphism may affect responses to a drug. Such information may also be useful for anticipation of possible resistance development. A total of 224 ion channel genes/subunits have been identified in the genome of D. immitis. Whole genome sequencing data of parasites from eight different geographical locations, four from ML-susceptible populations and the other four from ML-loss of efficacy (LOE) populations, were used for polymorphism analysis. We identified 1762 single nucleotide polymorphic (SNP) sites (1508 intronic and 126 exonic) in these 224 ion channel genes/subunits with an overall polymorphic rate of 0.18%. Of the SNPs found in the exon regions, 129 of them caused a non-synonymous type of polymorphism. Fourteen of the exonic SNPs caused a change in predicted secondary structure. A few of the SNPs identified may have an effect on gene expression, function of the protein and resistance selection processes., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite., (Copyright © 2016. Published by Elsevier Ltd.)

Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To clone and express the partial fragment of Csnk2b gene of Dirofilaria immitis in prokaryotic cells, and analyze the immunoreactivity., Methods: The partial fragment of Csnk2b gene was amplified by PCR with a pair of specific primers. The PCR product was cloned into pMD18-T, and then sub-cloned to pGEX-4T-1 expression vector. The constructed plasmid pGEX-4T-1-Csnk2b was transformed into E. coli Rosetta (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE and identified by Western blotting., Results: The PCR product was about 700 bp. Enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1-Csnk2b was constructed. SDS-PAGE results showed that the relative molecular weight (M(r)) of the fusion protein (GST-Csnk2b) was about 45 000. GST-Csnk2b reacted positively with mouse anti-D. immitis serum., Conclusion: The partial Csnk2b gene has been expressed in prokaryotic expression system and shows immunoreactivity.

Drug treatments for heartworm disease have not changed significantly in the last decade. Due to concerns about possible drug resistance and their lower efficacy against adult worms, there is a need for the development of new antifilarial drug therapies. The recent availability of genomic sequences for the related filarial parasite Brugia malayi and its Wolbachia endosymbiont enables genome-wide searching for new drug targets. Phosphoglycerate mutase (PGM) enzymes catalyze the critical isomerization of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG) in glycolytic and gluconeogenic metabolic pathways. There are two unrelated PGM enzymes, which are structurally distinct and possess different mechanisms of action. The mammalian enzyme requires 2,3-bisphosphoglycerate as a cofactor (dependent PGM or dPGM), while the other type of PGM does not (independent PGM or iPGM). In the present study, we have determined that Dirofilaria immitis and its Wolbachia endosymbiont both possess active iPGM. We describe the molecular characterization and catalytic properties of each enzyme. Our results will facilitate the discovery of selective inhibitors of these iPGMs as potentially novel drug treatments for heartworm disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We recently reported the cDNA cloning and functional characterization of a novel transglutaminase (TGase) from the dog filarial parasite Dirofilaria immitis. D. immitis TGase (DiTG) has no sequence similarity with any other known TGase, but has significant similarity to protein disulfide isomerase (PDI)-related endoplasmic reticulum protein ERp60. In the present study. we further characterized the recombinant DiTG (rDiTG) and studied its role in the molting process of third-stage larvae. The enzymatic activity of rDiTG requires Ca2+, and the maximum activity was observed at a calcium concentration of 4 mM. Interestingly, the rDiTG was highly thermostable, with optimal activity observed at 55 degrees C, similar to that seen with the native enzyme. Dithiothreitol (DTT) was not essential for enzyme activity. In fact, rDiTG was more active in the absence of DTT. The known inhibitors of TGase, such as monodansylcadaverine (MDC), cystamine and iodoacetamide, inhibited the TGase activity, but not the PDI activity, of rDiTG, demonstrating the dual activity of rDiTG. The TGase-specific pseudosubstrate, MDC, completely inhibited the molting of D. immitis L3 to L4 if present during the first 24-48 h of the molting process. Electron microscopic studies revealed that MDC-treated infective larvae failed to show separation between the L3 and L4 cuticles. The L4 cuticle and accompanying hypodermis were much thinner in MDC-treated worms than in controls. Using anti-rDiTG antiserum, the native DiTG antigen was localized in the hypodermis, afibrillar muscle cells and gut epithelium in adult male and female worms as well as developing embryos in the females.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.

A novel secreted aromatic amino acid decarboxylase-like molecule was identified in the excretory/secretory products of L3/L4 larvae as well as in an extract of adult Dirofilaria immitis. The secretion of the enzyme was developmentally regulated. Peak enzyme activities were detected in the culture medium before and after the molting of L3 larvae in vitro. The enzyme was purified from D. immitis adult extracts and the excretory/secretory products of L3/L4 larvae using different chromatographic methods followed by isoelectric focusing and SDS-PAGE. The enzyme has a molecular mass of 48 kDa and a pI of 5.6, and shows a specific enzymatic activity towards the aromatic amino acid substrates phenylalanine, tyrosine and tryptophan. The enzyme's activity did not show an absolute requirement for exogenous pyridoxal-5-phosphate. However, addition of pyridoxal-5-phosphate at 5 microM in the reaction increased the enzyme activity greatly. The enzyme had the ability to catalyze the formation of dopamine from L-dopa. Studies on the effects of inhibitors on the enzyme activity showed that the enzyme was sensitive to Pefabloc and p-chloromercuribenzoic acid, but not to diisopropyl flurophosphate. The Km values of the enzyme for H-Phe-AMC, H-Tyr-AMC and H-Trp-AMC were calculated to be 32.1 microM, 35.1 microM and 29.1 microM, respectively.

Fragments of putative chitin synthase (chs) genes from two filarial species (Brugia malayi and Dirofilaria immitis) were amplified by PCR using degenerate primers. The full genomic and cDNA sequences were obtained for the B. malayi chs gene (Bm-chs-1); the predicted amino acid sequence is highly similar, over a large region, to two CHS sequences of the nematode Caenorhabditis elegans and also to two insect CHS sequences. Bm-chs-1 is abundantly transcribed in B. malayi adult females, independent of their fertilization status, but is also expressed in males and microfilariae. Oocytes and early embryos contain large amounts of Bm-chs-1 transcript by in situ hybridization, but later stage embryos within the maternal uterus show little or no Bm-chs-1 transcript. No specific hybridization could be demonstrated in maternal somatic tissues. Polyclonal antibodies were raised against a peptide expressed from a recombinant cDNA fragment of Bm-chs-1; immunostaining detected CHS protein in oocytes and early to midstage embryos. These studies characterize a gene that is likely to be essential to oogenesis and embryonic development in a parasitic nematode. Because chitin synthesis and eggshell formation begin after fertilization, the presence of CHS protein in early oocytes suggests that the enzyme must be activated as a result of fertilization. These studies also demonstrate that chitin synthesis may not be restricted to eggshell formation in nematodes, as the Bm-chs-1 gene is transcribed in life cycle stages other than adult females.

Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells. However, the mechanisms of H2O2 removal by the filarial parasites are unclear. Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis. A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D. immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1). rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol. The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1). Consistent with the enzyme activity, D. immitis adult worms could detoxify exogenously added H2O2 in vitro. Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products. The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry. In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms. Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system.

The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells. Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities. In addition, the cuticle serves as a protective barrier against the host. A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles. One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced. The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids. GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level. Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia. Antibodies raised against D. immitis larval cuticles reacted with rDiAsp in immunoblots. This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode.

Microfilarial infections could be detected by the Difil Test in 11 (2.2%) of 479 blood samples of clinically asymptomatic dogs from the South of Switzerland. Dirofilaria repens and D. immitis were identified in 3 (0.6%) and 8 dogs (1.6%), respectively, by the acid phosphatase activity of the microfilariae. 10 dogs with microfilaremia had been abroad or a stay outside Switzerland could not be excluded. One dog diagnosed with D. immitis could have had acquired the infection in the canton Tessin according to information given by the owner. Dogs with microfilaremia are a potential source of infection for mosquitoes. An indigenous cycle of infection in the South of Switzerland is possible since the mean average temperature in summer is above 18 degrees C which is necessary for optimal parasite development in the vector. A strict control of imported dogs or animals exposed to the disease in endemic regions as well as the therapy of infected dogs in the South of Switzerland is advisable.

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase gene sequence and hybridization screening of D. immitis cDNA libraries. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site. The cDNA clone was expressed in Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively. An antiserum generated against the E. coli-expressed protein detected a protein of 29 kDa in D. immitis via immunoblotting. This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days of in vitro culture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.

The distribution of Dirofilaria immitis acid proteinase in adult worm tissue was examined biochemically and immunohistochemically. About 45% of the total proteinase activity of 700g supernatant, which was obtained from the 0.25 M sucrose homogenate of live adult worms, was found in the 100,000g supernatant by subcellular centrifugation analysis. The distribution pattern of the proteinase activity observed by Percoll density gradient centrifugation coincided with that of glucose-6-phosphatase, a marker cytosolic enzyme, suggesting that the acid proteinase was present in vivo in a membrane-free form, possibly in the cytosol or secretory fluid. Immunostaining for the proteinase in the parasite tissue using the IgG1 (kappa-type) monoclonal antibody, H-1, revealed immunoreactive enzyme primarily inside the cell as small grains, but not on the cell surface, and immunoreactivity was distributed widely in worm tissues such as lateral cords, dorsal and ventral median cords, the anterior end of the parasite, and intestinal epithelial cells in granular form. In the male reproductive system, the testicular wall and germ cells were labeled with the antibody, and in the female, uterine walls, fertilized eggs, and developing eggs as well as microfilaria were labeled. In conclusion, D. immitis acid proteinase is widely distributed in the parasite tissue, possibly functioning not only in nutrition metabolism but also in production of sperm and microfilariae, etc.

Mitochondrial complex II functions as a fumarate reductase (FRD), the reverse reaction of succinate dehydrogenase (SDH), and plays an important role in the anaerobic respiratory chain of parasitic helminths. In this study, complex II from the dog heartworm, Dirofilaria immitis adult, which is thought to act as a homolactatic fermenter, was examined in terms of its enzymatic features and primary structure in order to investigate the possible role of mitochondria in this filaria. Mitochondria from D. immitis adult showed high FRD activity when the enzymatic assay was performed using methylviologen as an artificial electron donor. The ratio of SDH to FRD in D. immitis was comparable to that in Ascaris suum adult, which is known to have an anaerobic mitochondrial respiratory chain with a high FRD activity of complex II. The FRD activity of D. immitis mitochondria was inhibited by the sulfhydryl reagent N-ethylmaleimide (NEM), while that of A. suum complex II was resistant to this inhibitor. The presence of the flavoprotein (Fp) subunit, which contains the substrate binding active site, was confirmed in D. immitis mitochondria by immunoblotting using a monoclonal antibody against the A. suum Fp subunit. By homology probing with the polymerase chain reaction, the entire cDNA for the D. immitis adult Fp was cloned and sequenced. The deduced amino acid sequence showed significant homology to that of A. suum and other mitochondrial Fps, in contrast to much less similarity to bacterial FRD, even though the D. immitis complex II showed high FRD activity. These results are the first indication of the presence of a functional complex II in D. immitis mitochondria.

An acid proteinase of Dirofilaria immitis worms was purified 437-fold by gel filtration on Sephadex G-75 followed by pepstatin-Agarose gel affinity chromatography. The enzyme with a molecular weight of 42 kDa was homogeneous as judged by both affinity chromatography and SDS-polyacrylamide gel electrophoresis. Polyacrylamide disc electrophoresis at pH 8.9, however, revealed that the enzyme was composed of five multi-forms, all carrying proteinase activity. Optimum pH of the enzyme was in the range of pH 2.8 to 3.4, and its isoelectric point ranged between 5.8 and 6.4. The purified proteinase showed a potent activity against hemoglobin and myoglobin releasing acid soluble peptides, but not free amino acids. When enzymatic properties of the proteinase was compared with mammalian cathepsin D and pepsin, D. immitis proteinase activity was reduced to about 80% of the initial activity by incubating at neutral pH and 50 degrees C for 5 min, just like cathepsin D, which remained intact. Pepsin activity was completely destroyed under the same condition. An aspartic proteinase inhibitor, 1,2-epoxy-3-(p-nitrophenoxy)propane, which inhibited pepsin by 30% at 37 degrees C for 10 min, did show little effects on D. immitis proteinase and cathepsin D. Inhibitory effect of diazoacetyl-DL-norleucine methyl ester (DAN) on D. immitis proteinase was intermediate (50% after 60 min). Immunolocalization of the proteinase in the worm tissue using its monoclonal antibodies revealed that the enzyme was localized in the intestine as well as uterine wall and some small granules of microfilariae in the uterus.

A superoxide dismutase (SOD) from adult worms of Dirofilaria immitis was purified using ethanol-chloroform and acetone treatment, DE 52 cellulose, and Sephadex G-75 gel chromatography to obtain a 677-fold purification and a specific activity of 5483 units/mg of protein. The purified SOD was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and composed of two identical 18.5 kDa subunits. The purified SOD was inactivated completely by 3 mM potassium cyanide, and reduced to about 18% of the initial activity by incubation at 100 degrees C for 10 min and to 28% by treatment with 2% SDS. This means that D. immitis SOD differs markedly from the mammalian Cu/Zn SOD, although it is indeed a Cu/Zn SOD. Atomic absorption spectrometry revealed the presence of 0.80 mole of Cu and 0.78 mole of Zn per mole of subunit. The enzyme consisted of 22.6% acidic and 11.9% basic amino acids. No free amino acid was detected in the SOD by N-terminal amino acid sequencing.

Subtle decreases in racing performance have been noted in asymptomatic greyhounds with heartworm burdens insufficient to obstruct pulmonary outflow, suggesting that alternative mechanisms may be involved in the pathogenesis of canine heartworm disease. Endothelium-dependent relaxation is depressed in the in vivo femoral artery of heartworm-infected dogs, in the in vitro pulmonary artery from heartworm-infected dogs, and in the in vitro rat aorta exposed to heartworms, heartworm-conditioned medium, and serum from heartworm-infected dogs. These findings suggest that circulating filarial factors may play a role in the pathogenesis of heartworm infection. We examined the effect of Dirofilaria immitis, the canine heartworm, on acetylcholine-induced contraction of rat tracheal rings. In epithelium-intact rings, both heartworms and heartworm-conditioned medium increased acetylcholine-induced contraction. Pretreatment of the parasites with aspirin prevented the filarial-induced increase in acetylcholine-induced contraction, suggesting that parasite cyclooxygenase products are responsible for the effect. In addition, heartworms caused contraction in both epithelium-intact and denuded rings and this effect was markedly decreased by pretreatment of the worms with aspirin. Filarial cyclooxygenase metabolites may cause airway hyperreactivity and parasite-derived factors could play a role in the subtle changes in exercise performance seen in asymptomatic greyhounds with low worm burdens. Comparable mechanisms may be operating in other filarial diseases, including those that affect humans in tropical countries.

D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By Day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl-Phe-Ala-CH2F (Z-Phe-Ala-CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concentrations of inhibitor more larvae had initiated, but not completed, the molt. In addition to Z-Phe-Ala-CH2F, four other fluoromethyl ketone derivatives, Z-Phe-Arg-CH2F, amorpholine urea-(Mu)-Leu-Phe-CH2F, Mu-Tyr-Phe-CH2F, and Mu-Phe-Phe-CH2F, were tested to determine their effects on L3 in culture. All fluoromethyl ketones tested except Z-Phe-Arg-CH2F inhibited molting. Larvae cultured in inhibitors were determined to be alive as judged qualitatively by motility and quantitatively by reduction of 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone derivative had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone derivative that did not inhibit molting, Z-Phe-Arg-CH2F, was a slightly less effective inhibitor of larval extract-initiated hydrolysis of the synthetic peptide substrate, Z-Val-Leu-Arg-7-amino-4-methyl coumarin. L3 were also cultured through the molt in media containing the synthetic peptide substrate Z-Val-Leu-Arg-4- methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fluorescence as seen on Days 0-4 during the molting process was first observed on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle.

An acid proteinase of Dirofilaria immitis worms was purified 437-fold by gel filtration on Sephadex G-75 followed by pepstatin-agarose gel affinity chromatography. The enzyme with a molecular weight of 42,000 and specific activity of 384 units (mg protein)-1 was homogeneous as judged by both affinity chromatography and SDS-polyacrylamide gel electrophoresis. However, polyacrylamide disc electrophoresis at pH 8.9 revealed that the enzyme is composed of 5 multiforms, all carrying proteinase activity. Optimum pH of the enzyme was in the range of pH 2.8-3.4, and its isoelectric point ranged between 5.8 and 6.4. The purified proteinase showed a potent activity against hemoglobin and myoglobin releasing acid soluble peptides, but not free amino acids. Complete inhibition of the proteolytic activity by pepstatin in the order of 10(-7) M strongly suggests that the enzyme belongs to the carboxyl-proteinase family.

Superoxide dismutase (SOD) may not only perform a housekeeping role in filarial worms but also assist in defense against oxidants generated by host immune cells. Both Dirofilaria and Onchocerca adult filariae and microfilariae contain relatively high activities of the antioxidant enzyme SOD; adult Dirofilaria worms also secrete SOD in vitro. In addition, superoxide radicals are relatively impotent against Dirofilaria and Onchocerca microfilariae in vitro. In assessing the role of SOD, we determined the anatomic localization of SOD in D. immitis adult worms by immunolocalization at the light-microscopic level. We found that anti-D. immitis SOD did not stain parasite tissues homogeneously, in support of the hypothesis that SOD does not have only a housekeeping role and that the pattern of staining may suggest another role(s) for SOD.

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.

Superoxide dismutase (SOD) was purified to apparent homogeneity from Dirofilaria immitis, the causative agent of Dog Heartworm disease which is prevalent in the Southeastern United States. The enzyme has a molecular weight of 18,000 under denaturing conditions with an isoelectric point of 5.6. Both values are similar to those found for previously purified helminth SODs. The amino acid analysis shows greater similarity with mammalian SODs than with the published Schistosoma mansoni SOD, probably because the S. mansoni enzyme appears to be an extracellular, not a cytosolic, SOD. Although SOD activity is easily detected in D. immitis homogenates, the hydrogen peroxide scavenging activities of catalase and glutathione peroxidase were below the limits of our assay. This suggests that D. immitis primary defense against oxidants may be SOD. We feel that this line of research may provide valuable insights into a vulnerable area of D. immitis that may be a good target for drug therapy.

The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis. The level of Fru-2,6-P2 was determined to be 4 +/- 0.3 nmol(g wet weight)-1 in the tissues of the filariid. Fru-2,6-P2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The Kact values for Fru-2,6-P2 were 378 +/- 18 nM and 65 +/- 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8. AMP also stimulated the activity of both forms of the enzyme with Kact values of 230 +/- 10 microM and 37.3 +/- 6.1 microM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the KFru-6-P values were 153 microM and 60 microM for the non-phosphorylated and phosphorylated forms of the PFK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence has been presented showing two kinds of acidic protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum. A haemoglobinolytic activity without adding any SH-containing compounds was maximal at pH 3.5, 2.5, 3.0 and 3.5 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The inhibitor studies demonstrated that this activity is ascribable to carboxyl protease(s). In the presence of dithiothreitol, activity on Azocoll (azo-dye coupled hide powder) was maximal at pH 4.6, 4.6, 3.5 and 5.6 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The effects of inhibitors demonstrated that this activity belongs to the thiol protease class. The intraspecific distribution of the protease activities was studied in some of the nematodes from which the organs could be anatomically separated. The distribution patterns of the haemoglobinolytic and azocollytic activities were quite different in An. cantonensis and much the same in As. suum. Based on the present results, acidic haemoglobinolytic activities reported in adult S. mansoni by other authors are thought to be due to carboxyl and thiol protease(s) respectively.

Glutathione S-transferase (EC 2.5.1.18) was detected in the cytosolic and microsomal fractions of adult Dirofilaria immitis females at respective levels of 30 nmol and 3 nmol min-1 (mg protein)-1 activity with the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The transferase activity in the cytosolic fraction of adult Brugia pahangi females was 10 nmol min-1 mg-1 with CDNB; determination of its activity in the microsomal fraction of this filariid was not attempted. These filarial glutathione S-transferases were further characterized after their purification by glutathione-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytosolic transferase from D. immitis, molecular weight 47000, yielded a single subunit of around 28 kDa. The cytosolic and microsomal transferases from D. immitis differed in their activity with CDNB, 1,2-dichloro-4-nitrobenzene, 4-benzylchloride and ethacrynic acid. The cytosolic transferase from B. pahangi was distinguished by its high activity with ethacrynic acid. Both glutathione S-transferases from D. immitis also functioned as a glutathione peroxidase, strongly preferring cumene hydroperoxide as a substrate over hydrogen peroxide. Both were equiactive inhibitors of malonaldehyde formation in the NADPH-microsomal lipid peroxidation system. Thus, in addition to the ability of filarial glutathione S-transferases to detoxify electrophilic xenobiotics, at least those from D. immitis also exhibited selenium-independent glutathione peroxidase activity. Their glutathione S-transferase function suggests a potential role for these enzymes in the leukotriene synthetic pathway, if filariae can form such eicosanoids from arachidonate. Functioning as a glutathione peroxidase, they could serve to protect filarial membrane lipids from peroxidation.

1. Dirofilaria immitis hexokinase was relatively heat stable and had a pH optimum range between 7.8 and 8.2. 2. Mean Vmax was 0.40 +/- 0.10 (S.D.) mumole/min/100 mg of worm. 3. Mean Km values were 0.32 mM for glucose, 0.86 mM for fructose and 0.39 mM for ATP. 4. Glucose-6-phosphate was not a strong product inhibitor. 5. Starch gel electrophoresis demonstrated at least three isozymes.

A multilocus genetic analysis based on protein electrophoresis was carried out on 39 adult specimens of Dirofilaria repens and 31 of D. immitis. Seventeen enzymatic loci provided reliable electrophoretic patterns which were compared in the two species. All loci except one were monomorphic. Fixed alternative allozymes were found at 13 loci while only 3 loci shared apparently the same allozyme in the two species. The polymorphic locus, Pgm, also showed alternative allozymes. This remarkably high genetic divergence, which presumably reflects a very old speciation process, allows an easy characterization of D. repens and D. immitis and supports their classification in the subgenera Nochtiella and Dirofilaria, respectively.

Two acid proteases were isolated from the soluble extracts of adult Dirofilaria immitis, the filarial heartworm of canines. Activity of these proteases was detected using 3H-labeled bovine alpha-casein as substrate, and they were designated Fp-I and Fp-II in order of their elution from a CM-cellulose column. The molecular weight of partially purified Fp-I was approximately 170000, and it was active between pH 4.6-5.8. The activity of Fp-I doubled in the presence of various sulfhydryl reagents at 5 mM, and it was inhibited 50-60% by the sulfhydryl inhibitors p-hydroxymercuribenzoate and iodoacetate at 1 mM, the heavy metal chelating agent o-phenanthroline at 1 mM and the peptide aldehyde protease inhibitors pepstatin (10 microM), leupeptin, antipain and chymostatin (50 microM). The molecular weight of the more extensively purified Fp-II is approximately 48000. This protease was active between pH 2.6-3.4 and was highly sensitive to inhibition by pepstatin (80% inhibition at 10 nM). Fp-II was not significantly affected by sulfhydryl reagents, sulfhydryl inhibitors, metal chelating agents or peptide aldehyde protease inhibitors other than pepstatin. These properties of dirofilarial Fp-II resemble those of mammalian cathepsin D.

The presence of 5'-nucleotidase was demonstrated in Onchocerca volvulus and Dirofilaria immitis; the bulk of activity was found in the particulate fraction. The enzyme of filarial worms exhibited a broad pH-optimum between 6.4 and 8.0 and substrate specificity for nucleotides compared to glucose-6-phosphate and p-nitrophenyl phosphate. The apparent Km-values for AMP were found to be 0.15 mM and 0.22 mM for the enzyme from O. volvulus and D. immitis, respectively. The activity of 5'-nucleotidase from both filarial worms was effectively inhibited by the filaricidal compound CGP 8065, a dithiocarbamate-derivative of amoscanate, whereas the 5'-nucleotidase from rat liver was not affected. The parasite-specific inhibition by CGP 8065 was found to be reversible and to be competitive with respect to the substrate AMP. The inhibition constants were calculated to be 24 microM and 8 microM for the enzyme from O. volvulus and D. immitis, respectively.

The histochemical distribution of acid phosphatase in microfilariae and in the larval stages of four mosquito-borne filariae: Wuchereria bancrofti, Brugia malayi, B. pahangi and Dirofilaria immitis was studied using naphthol AS-TR-hexazonium technique and light microscopy. Accurate differentiation between microfilariae of the four species could be made on the basis of their patterns of acid phosphatase activity. In contrast to microfilariae in the blood, the larval stages in the mosquito exhibited different patterns of acid phosphatase activity which were characteristic for each developmental stage. In the first-stage larva, maximum acid phosphatase activity was found in the anal vesicle, the growing anal membrane (anal plug), buccal cavity, forming intestine and rectum. In the second-stage larva, acid phosphatase activity was present throughout the alimentary canal, particularly in the section of the intestine and rectum. In the infective third-stage larva, the whole body stained densely red. The reaction for acid phosphatase in the excretory cell complex of W. bancrofti and of both species of Brugia gradually decreased in intensity and disappeared completely towards the end of the first-larval stage, whereas in D. immitis a strong reaction in this area persisted throughout the larval life in the mosquito. The presence or absence of enzymic activity in the excretory cell complex and in the Mundgebilde (amphids) of the developing larvae can be used as an adjunctive diagnostic method.

Adult Dirofilaria immitis and Brugia pahangi were found to possess the following folate-related enzymes that catalyze the formation of 5,10-methenyltetrahydrofolate (methenylFH4) or 10-formylFH4 (f10FH4): f10FH4 synthetase, methenylFH4 cyclohydrolase, f5FH4 cyclodehydrase, and a bifunctional complex composed of formiminoglutamate: FH4 formiminotransferase and 5-fomiminoFH4 cyclodeaminase. The properties of these filarial enzymes were generally similar to those of their counterparts from invertebrate and vertebrate sources, although each possessed one or more distinctive characteristics.

A potent inhibition of arginyl-tRNA-synthetase from Dirofilaria immitis by N-[4-(4'nitroanilino)-phenyl]-S-(beta-carboxylethyl)-dith iocarbamic acid-ester (CGP 8065) is demonstrated, which indicates the charging of amino acids to tRNA as a possible target for the chemotherapeutic attack by this filaricidal compound. CGP 8065 competes with ATP; the inhibition constant was determined to be 13 micrograms ml-1 (34 microM). The Michaelis constants for arginine and ATP were found to be 5 microM and 0.4 mM, respectively. The inhibition of arginyl-tRNA-synthetase by CGP 8065 was shown to be specific for D. immitis; the isofunctional enzymes from Ascaris suum and rat liver were not affected.

The biologically active sulfidopeptide leukotriene, leukotriene C4, is formed by the enzymatic action of leukotriene C4 synthase, which conjugates glutathione with leukotriene A4. We have found that a filarial glutathione S-transferase can function as a leukotriene C4 synthase. Glutathione S-transferase was purified from the cytosol of adult Dirofilaria immitis by glutathione-agarose affinity chromatography and was reacted with 25 microM leukotriene A4 methyl ester and 10 mM glutathione. The filarial enzyme catalyzed the formation of leukotriene C4 methyl ester, as shown by reverse phase high pressure liquid chromatographic analyses. The finding that filarial glutathione S-transferase can function as leukotriene C4 synthase provides a mechanism whereby filarial parasites could form lipoxygenase pathway derived sulfidopeptide leukotrienes.

Adult Brugia pahangi and Dirofilaria immitis were found to possess the following four enzymes that are associated with the cofactor 5,10-methylenetetrahydrofolate (CH2FH4): serine hydroxymethyltransferase, thymidylate synthetase, CH2FH4 dehydrogenase, and CH2FH4 reductase. The properties of the isoenzymes from the two filariae were virtually indistinguishable, except that diethylcarbamazine inhibited CH2FH4 reductase from B. pahangi 50% at 10 muM, but did not not affect the isoenzyme from D. immitis at 100 muM. The properties of these four filarial enzymes generally were similar to their counterparts from mosquitoes and mammalian sources, but several notable differences were identified.

Proteases were detected in aqueous extracts of Dirofilaria immitis microfilariae. Enzymes within the extract were capable of hydrolyzing Azocoll, a general protease substrate, at pH's 7, 8, and 9. Sensitivities to a variety of protease inhibitors indicated that multiple azocollytic enzymes were present in the extract, most prominent of which appear to belong to the serine class of proteases. By incorporating various substrates into the matrices of polyacrylamide gels, 2 SDS-resistant, mercaptoethanol-sensitive proteases in the MF extract were identified at 22 and 76 kDa. These proteases showed differential abilities to digest casein, fibrinogen, hemoglobin, and IgG. The MF extract hydrolyzed radiolabeled IgG into 8-10-kDa fragments following a 20-hr incubation. A similar degree of digestion was observed in 2 hr when viable microfilariae were used. The potential significance of these proteases in the evasion of host effector mechanisms is discussed.

Anti-acetylcholinesterase activity of diethylcarbamazine (DEC) was studied. Acethylcholinesterase (AchE) of adult worms of Dirofilaria immitis, those of the 4th-stage larvae, early 5th-stage larvae and adults worms of Brugia pahangi, and that of hamster brain tissue were all inhibited by DEC. Michaelis constant (Km) of D. immitis and B. pahangi adult worm AchE were 1.47 x 10(-4) and 1.81 x 10(-4) M respectively. DEC was a competitive inhibitor of the filarial AchE. Inhibition constant (Ki) for AchE of D. immitis and B. pahangi adult worms were 2.56 x 10(-4) and 6.39 x 10(-4) M, respectively. DEC is a less potent anticholinesterase inhibitor, because Ki of DEC is 10(4) times higher than that of eserine, a potent anti-cholinesterase agent.

Lactate dehydrogenase from adult D. immitis was partially purified and characterized. The molecular weight of the enzyme was determined to be 130 000. The specific activity of the lactate dehydrogenase was 4.1 U/mg in the direction of NADH-oxidation and 0.38 U/mg protein in the direction of NAD-reduction. The Michaelis constants were determined to be 0.028 mM and 0.9 mM for NADH and NAD, respectively. Suramin was found to be a potent inhibitor of the lactate dehydrogenase activity. The inhibition constant was calculated to be 0.006 mM. The type of inhibition by suramin was competitive with respect to the cofactors. The Km-values for pyruvate and lactate were determined to be 0.3 mM and 11mM, respectively.

Homogenates of adult Dirofilaria immitis possess a microsomal enzyme system able to transfer mannose from GDPmannose to endogenous lipid intermediate(s) and exogenous dolichol monophosphate. A divalent metal was required with Mn2+ being the most effective; other requirements for optimal activity included Triton X-100, EDTA and either ATP or NaF. The maximal rate of mannose transfer to the lipid acceptor by the filarial system, 1.6 pmol.min-1.mg-1 protein, occurred at 37 degrees C and pH 7.0, and this was inhibited 50% by 8 microM diumycin and not at all by 100 microM tunicamycin. D. immitis microsomes also were shown to promote the transfer of mannose to derivatives of alpha-lactalbumin, resulting in the synthesis of a mannose-labeled glycoprotein.

Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.

NADP-linked malic enzyme (malate dehydrogenase (oxaloacetate-decarboxylating) NADP+, EC 1.1.1.40) has been partially purified from adult Onchocerca volvulus and Dirofilaria immitis. Suramin was found to inhibit the activity of malic enzyme from both filarial worms. The inhibition constants for suramin were calculated to be 0.011 microM and 0.015 microM for the enzymes from O. volvulus and D. immitis, respectively. In the case of NADP-linked malic enzyme from Trypanosoma brucei and chicken liver the inhibition by suramin was less pronounced. The inhibition constants were found to be 0.8 microM and 2.5 microM for the protozoan and vertebrate enzymes, respectively. The type of inhibition was competitive with respect to malate. The Michaelis constants for malate and pyruvate were determined to be 0.9 and 4.5 mM for O. volvulus and 0.85 and 5.0 mM for D. immitis, respectively. The low Km values for malate compared to those for pyruvate and the about 15-fold greater turnover in the direction of decarboxylation compared to carboxylation indicated that malic enzyme from both filarial sources might be involved in an alternative pathway leading from phosphoenolpyruvate via oxaleacetate, malate and pyruvate to lactate. It is suggested, that the inhibition of malic enzyme activity from O. volvulus by suramin might interfere with the generation of NADPH for biosynthetic reactions.